Clonal dissemination of Staphylococcus aureus isolates causing nosocomial infections, Tehran, Iran

Objective(s): In the current research, the prevalence of Staphylococcus aureus clones and genes encoding antimicrobial resistance and toxins were examined among 120 S. aureus strains from nosocomial infections in tehran, Iran. Materials and Methods: Antimicrobial susceptibility was examined, based on disk diffusion and PCR method to identify resistance and toxin-encoding genes. Based on the polymorphisms in SCCmec, agr, spa, and MLST, the isolates were typed. Results: Among 120 S. aureus isolates, 85 (70.8%) were methicilin resistant S. aureus (MRSA), and 35 (29.2%) were methicilin sensetive S. aureus (MSSA). The tested isolates contained resistance genes, including ant(4΄)-Ia (90%), aac(6΄)-Ie/aph(2˝) (80%), aph(3΄)-IIIa (30%), erm(A) (26.7%), erm(B) (10.8%), erm(C) (11.7%), msr(A) (40.8%), msr(B) (14.2%), tet(M) (45.8%), and mupA (8.3%). The MRSA strains were clustered into six different clones. The most common genotypes included ST239-SCCmec III/t037 (23.3%), ST239-SCCmec III/t388 (22.5%), ST22-SCCmec IV/t790 (8.3%), ST15-SCCmec IV/t084 (7.5%), ST585-SCCmec III/t713 (5%), and ST239-SCCmec III/t924 (4.2%), respectively. ST182/t196 (8.3%) and ST123/t171 (5%) belonged exclusively to MSSA strains. Overall, 10 (66.7%) and 5 (33.3%) out of 15 isolates with pvl genes were attributed to clones ST22-SCCmec IV/t790 and ST15-SCCmec IV/t084, respectively. ST22-SCCmec IV/t790, ST239-SCCmec III/t037, and ST15-SCCmec IV/t084, were related to high-level mupirocin-resistant phenotypes. Conclusion: The genetic diversity of S. aureus was confirmed in our hospitals, and ST239-SCCmec III/t037 showed a relatively high prevalence in our study. It seems that assessment of resistance and virulence genes in different S. aureus molecular types is necessary for proper antibiotic consumption.


Introduction
Staphylococcus aureus, which is described as a common nosocomial pathogen, is responsible for various diseases, such as food poisoning, osteomyelitis, wound infections, and even fatal conditions, such as endocarditis (1). Over the past few decades, it has been well-documented that the pathogen's resistance potential to antimicrobial agents, especially methicillin, may lead to its persistence in the hospital and community (2). In 1961, the first case of methicillinresistant S. aureus (MRSA) occurred in the UK (3). The prevalence of this infection has steadily increased since then, as confirmed in several studies, raising major concerns about the global increase in its prevalence, as well as its associated mortality and morbidity in the healthcare setting, especially intensive care units (ICUs) due to MRSA infections (1)(2)(3).
Resistance to methicillin is attributed to β-lactamase expression or changes in the structure of mecA geneencoded penicillin-binding protein-2. Generally, mecA gene (21-67 kbp) is recognized as a staphylococcal cassette chromosome mec (SCCmec). Generally, SCCmec is categorized into 11 types with respect to mec genes and ccr gene complexes (4). Identification of SCCmec type among S. aureus clinical isolates can be useful in molecular typing of MRSA strains (5).
Based on previous findings, SCCmec I-III and IV-V are respectively responsible for the most common hospital-acquired and community-acquired MRSA (HA-MRSA and CA-MRSA, respectively) infections. HA-and CA-MRSA strains can be distinguished with respect to some genotypic, phenotypic, and epidemiological characteristics, as well as virulence factors (4,5).
The emergence and prevalence of MRSA infections containing multidrug-resistant (MDR) genes have significantly limited the availability of antibiotics over the past decades. In addition, the growing emergence of MDR-MRSA strains poses a major global health concern (1). Wide resistance to β-lactams, besides other antibiotics, including aminoglycosides, lincosamides, and macrolides, has been shown in MRSA strains (6).
Aminoglycosides play a key role in serious antistaphylococcal therapies. According to the previous researches, resistance to aminoglycosides is attributed Molecular characterization of S. aureus clinical isolates Goudarzi et al. to aminoglycoside-modifying enzymes (AMEs) including aminoglycoside nucleotidyltransferases, aminoglycoside phosphotransferases and aminoglycoside acetyltransferases (7). mupA and mupB genes are responsible for resistance to mupirocin which is used to treat various types of skin diseases caused by S. aureus (8). Resistance to macrolides as protein synthesis inhibitors is mediated by msr genes activating efflux pumps and erm genes modifying the ribosomal binding site (9). Thus, in spite of new antibiotics introduction, concerning the emergence and dissemination of antibiotic resistance genes, MRSA infections treatment is still a great dilemma worldwide.
This study was conducted to identify antibiotic resistance patterns and the carriage of resistance and virulence genes as well as major MRSA clones by MLST, spa, SCCmec and agr techniques in clinical samples taken from patients in Tehran, Iran.

Sampling, MRSA isolation and antibacterial susceptibility testing
This cross-sectional study included 368 clinical samples from wound, blood, and urine specimens during April-December 2016. Ethics Committee of Shahid Beheshti University of Medical Sciences approved the implementation of this study (IR.SBMU. SM.REC.1395.157). All patients signed written informed consent forms.
Using E-test strips (bioMe´rieux), the minimum inhibitory concentrations (MICs) were measured for mupirocin and vancomycin. Resistance to three antibiotic groups or more, besides beta-lactams, was defined as MDR. High mupirocin resistance was defined as antibiotic use ≥ 256 mg/l. Growth in a well containing clindamycin and erythromycin (0.5 and 4 μg/ml, respectively) indicated inducible macrolide-lincosamidestreptogramin B and/or clindamycin resistance phenotypes; otherwise, constitutive MLSB and/or clindamycin resistance phenotype was confirmed (11). ATCC29213 and ATCC25923 (S. aureus) were considered as the reference strains for the quality control purposes.

Extraction of genomic DNA
For extracting genomic DNA, pure overnight S. aureus cultures were used on 5% sheep blood agar (BA; Merck, Germany), based on the protocols of InstaGene Matrix kit (BioRad, USA).

Multiplex PCR for SCCmec typing
According to a study by Boy and colleagues, for  (attributed to types I, II, III, IV, and V, respectively).

Multiplex PCR amplification for agr typing
In addition, for agr typing, multiplex PCR amplification was carried out, using forward (Pan) and reverse (agr1 to agr4) primers for the agr groups as previously recommended by Gilot et al (18). The specific oligonucleotide primers are listed in Table 1.

spa typing
On the other hand, spa gene was detected according to a study by Harmsen and colleagues (19). After the positive spa PCR products were purified, DNA sequencing was carried out in both strands (Macrogen; South Korea). Chromas 1.45 (Australia) was used to edit the sequences. To assign the sequences to specific spa types, the Ridom SpaServer database was searched.

MLST technique
Via amplification and sequencing, MLST was carried out on S. aureus isolates. The internal fragments of housekeeping genes were used to identify the allelic profiles; these genes included gmk, arcC, aroE, glpF, pta, yqiL, and tpi. The isolate was assigned a sequence type (ST) after comparing the sequences with the S. aureus MLST database.
None of the isolates were susceptible to all of the antimicrobial agents tested regarding in vitro antimicrobial susceptibility tests. All isolates were susceptible to vancomycin, among which 55 (45.8%), 48 (40%) and 17 (14.2%) isolates had a MIC of 0.5, 1 and 2 µg/ml, respectively.
Among the 35 MSSA isolates, no mupirocin resistance was detected, whereas 30 MRSA isolates (35.3%) were mupirocin resistant. Of these mupirocin resistant isolates, 14 (46.7%) and 16 (53.3%) had high and low resistance levels, respectively. All the highlevel mupirocin-resistant (HLMUPR) isolates were collected from wound samples. Patients diagnosed with mupirocin-susceptible and -resistant MRSA infections were not significantly different in terms of age and gender (P= 0.145 and 0.128, respectively). The frequency of resistance for MRSA and MSSA isolates to different antibacterial agents are presented in Table 2.
Of the 120 S. aureus isolates, 87.5% (105/120) were defined as MDR. The predominant multiple drug resistance profile among the MDR isolates were resistance to 9 and 7 antibiotics found in 75 (62.5%) and 12 (10%) isolates, respectively. Distribution of resistance profile and different clinical sample in S. aureus isolated from nosocomial infections are presented in Figure 1.

The distribution of resistance genes
In addition, the frequency of antibiotic resistance genes was measured. The genes included ant(4΄)-Ia

Distribution of SCCmec types
According to SCCmec typing, 66 (77.6%) and 19 (22.4%) MRSA isolates contained SCCmec types III and IV, respectively. No isolate harbored SCCmec type V, II, or I. Based on the multiplex PCR, isolates positive for PVL were attributed to SCCmec type IV, while tst gene was found in MRSA isolates from SCCmec III and IV. The HA-MRSA origin was emphasized by the presence of SCCmec type III.

Discussion
In consistent with the results of a multicenter study made by Ko et al. (20), a relatively high resistance to gentamicin (75%), amikacin (68.3%), kanamycin (77.5%), and tobramycin (59.2%) was reported in this study which was higher than the resistance rate reported by Goudarzi et al (12). In contrast to the findings of Marghaki et al `s study (6) who reported high frequency of aac(6′)/aph(2′′) gene (40.3% ) in comparison with other AME genes, in the present study, ant(4΄)-Ia (90%) was the most frequent AME gene in S. aureus isolates. However, in this study, aph(3΄)-IIIa gene frequency rate was higher than the study reported by Ida et al. Mupirocin as an important agent in the control of MRSA outbreaks and eradicating MRSA colonization was used for the treatment of different types of staphylococcal skin infections. Long period widespread use of mupirocin may lead to the emergence of mupirocin resistance S. aureus strains (8). In this survey, 30 isolates (25%) presented mupirocin resistance phenotype and 14 (11.7%) isolates were confirmed as HLMUPR-MRSA which is relatively lower than study in Iran (40%) (22) and higher than study in India (5%) (23) and Jordan (2.6%) (24). However, as a result of proper mupirocin prescription in clinic, in our study mupirocin resistant S. aureus isolates were found to be lower. We detected the resistance gene mupA in 10 isolates (8.3%) which is lower than 12.6% (25) and 25% in Iran (22).
In accordance with the results of our previous study (25) a high tetracycline resistance was seen in the present work (70%). In consistent with others (25), in our study the tet(M) gene that may cause tetracycline resistance was detected in 55 (45.8%) isolates.
In this study, 58 isolates (48.3%) presented cMLSB phenotype while the frequency of iMLSB phenotype was 10% (12/120). In consistent with our findings, Schreckenberger et al. (7%) reported low frequency of iMLSB phenotype among S. aureus isolates as well (26). According to the literature, there are discrepant rates of inducible clindamycin resistance in different geographic area. Rashid Nezhad et al. performed a study in seven Iranian teaching hospitals (25). They found that the frequency of cMLSB, iMLSB and MLS B phenotypes was 52.6%, 12.6%, and 5.3% respectively. Similarly, Fiebelkorn et al. in USA (27) reported that of 114 S. aureus isolates resistance to erythromycin, 34% and 29% indicated constitutive and inducible resistance pattern respectively. In a Canadian survey (28), the frequency of iMLSB and cMLSB phenotypes was found to be 64.7% and 35.3% respectively. In current work, the frequency of constitutive resistance was found to be higher than inducible resistance which is in line with the findings reported by Rashidi Nezhad et al (25).
Type III was the most frequently found SCCmec (77.6%), according to the results of SCCmec typing, associated with an MDR pattern among MRSA isolates. This is in consistence with the results reported by Japoni and colleagues (10). The nosocomial origin of the samples was confirmed by the high frequency of SCCmec type III.
A significant relationship was found between the expression of virulence factors and specific agr locus. In consistence with our previous study, the most common agr types were agr type I (75.8%), type III (16.7%), and type II (7.5%), respectively. Goudarzi et al. (12) reported agr type III to be the most frequent type of SCCmec in Iran. According to several studies, the frequency of toxin and adhesion molecules was higher in isolates harboring agr type I gene in comparison with those harboring agr type III; this is in agreement with our study. Therefore, it can presume that regulation of staphylococcal adhesion molecules and toxins is associated with agr type I.
According to the spa typing results, spa t037 was recognized as the most common spa type (23.3%). This spa type was reported from Saudi Arabia, China, Iran as well as among HA-MRSA isolates found in Europe, America and other regions of Asia (12,29,30).
In present study spa type t388 was estimated at 22.5%. This spa type has also been reported in a study in Iran (31). Similarly, in a study performed in Taiwan, Ho et al described spa type t388 in MRSA strains recovered from blood cultures in different medical centers (32). It seems that the prevalence of t388 is progressively increased and has been successfully established in our healthcare settings. In our study, the frequency of t713 and t924, earlier reported in UAE and Iran (12), were found to be 16.8% and 8.3% respectively.
In contrast with previous study conducted in Iran (12), in this study, low frequency of t790 and t084 spa types among our isolates, was also demonstrated. For the first time we are reporting t196, and t171 spa types in MSSA strains from Iran.
Using various MRSA typing methods, the isolates were attributed to six different clones, namely ST239-SCCmec III/t388, ST22-SCCmec IV/t790, ST239-SCCmec III/t037, ST15-SCCmec IV/t084, ST585-SCCmec III/ t713, and ST239-SCCmec III/t924. In line with previous study from Iran (12), ST239-SCCmec III/t037 clone is currently more frequent in our hospitals (23.3%). The review of the literature reveals that the multiresistant ST239 clone is responsible for at least 90% of HA-MRSA infections in Europe, United States, and some Asian countries, including Kuwait and Malaysia (29). Therefore, the presence of ST239-SCCmec III/t037 in our healthcare setting might be attributed to neighboring regions.
ST239-SCCmec III/t388 (22.5%) was the second most commonly detected MRSA clone. A similar result was reported by Ohadian Moghadam et al from Iran in which major universal MRSA clones were described as ST239, ST291, and ST30 (31).
Based on the findings, in clinical MRSA strains, ST15-SCCmec IV/t084 (7.5%) was the fourth most commonly detected clone. The low frequency of this clone has been previously reported in 16 European countries (36). PVL-carrying ST15 isolates were identified in a study by Rasigade and colleagues on 211 S. aureus strains from 19 different countries (37). Five (4.2%) ST15-SCCmec IV/ t084 isolates carried pvl genes in our study. Previously, PVL-positive ST15 was reported by Japoni-Nejad and colleagues in Iran (10). In contrast to several studies in which ST15 was reported to be prevalent among CA-and HA-MSSA isolates, all ST15 isolates belonged to MRSA strains in our study. We reported the presence of ST585-SCCmec III/t713 in 5% of isolates. In another study by Goudarzi et al the molecular features of MRSA isolates were identified, and ST585-SCCmec III/t713 was reported in 12% of blood samples from bacteremia patients (12).
To sum up, the present findings showed that MRSA isolates have various genetic backgrounds in our hospitals and involve six major clones. Certain molecular types were associated with some resistance and virulence genes (e.g., eta with ST22-SCCmec IV/t790, ST15-SCCmec IV/t084, and ST239-SCCmec III/t037; mupA with ST15-SCCmec IV/t084 and ST22-SCCmec IV/ t790; pvl with ST15-SCCmec IV/t084 and ST22-SCCmec IV/t790; etb with ST585-SCCmec III/t713 and ST22-SCCmec IV/t790). The presence of eight different spa types, i.e., t037, t388, t713, t924, t790, t196, t084, and t171, was also confirmed. For the first time in Iran, STs 182 and 123, as well as spa types t196 and t171, were detected, which might be indicative of the emergence of new clones. Further studies on other neighboring regions, focusing on the emergence of new circulating clones, are necessary to reach an overall understanding of dynamic MRSA clones in Iran and the Middle East.